MARCO CONCEPTUAL

Human PXR Forms a Tryptophan Zipper-Mediated Homodimer†

Reproduced with permission from Biochemistry (2006) 45: 8579-89 Copyright 2006 American Chemical Society.

Schroeder M. Noble‡_{, Virginia E. Carnahan}‡_{, Linda B. Moore}§_{, Tom Luntz}||_{, Hongbing}

Wang||, Olivia R. Ittoop§, Julie B. Stimmel§, Paula R. Davis-Searles+, Ryan E. Watkins‡, G. Bruce Wisely§, Ed LeCluyse||, Ashutosh Tripathy‡, Donald P. McDonnell⊥ and

Matthew R. Redinbo+, ‡,*

‡_{Department of Biochemistry and Biophysics, University of North Carolina at Chapel}

Hill, Chapel Hill, NC 27599

§_{Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, NC}

27709

||_{Division of Drug Discovery and Disposition, School of Pharmacy, University of North}

Carolina at Chapel Hill, Chapel Hill, NC 27599

⊥_{Department of Pharmacology and Cancer Biology, Duke University Medical Center,} Durham, NC 27710

+_{Department of Chemistry, and the Lineberger Comprehensive Cancer Center, University}

of North Carolina at Chapel Hill, Chapel Hill, NC 27599

†_{This work was supported by NIH grant DK62229 (M.R.R.), NIH ATLAS grant}

DK62434 (D.P.M.), and a National Science Foundation Graduate Research Fellowship (V.E.C.).

*_{Corresponding Author: Department of Chemistry, Campus Box #3290, University of}

This manuscript was a collaborative effort. I performed the mammalian two-hybrid studies.

Abstract

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal β-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface β-strands, generating a ten-stranded intermolecular β-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR’s ability to interact with DNA, RXRα, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor’s recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor.

Introduction

The human pregnane X receptor PXR plays a important role in controlling the expression of genes central to drug and endobiotic metabolism, including those encoding cytochrome P450s (CYPs), UDP-glucuronosyl-transferases, glutathione-S-transferases, and drug efflux pumps (Gardner-Stephen, D., et al. 2004; Geick, A., et al. 2001; Gerbal- Chaloin, S., et al. 2002; Kliewer, S.A. 2003; Xie, W., et al. 2001a). PXR is considered to be a master regulator of the expression of CYP 3A4 isoform, which metabolizes more than 50% of human drugs (Maurel, P. 1996). PXR is expressed largely in the liver and intestines and responds to a wide variety of structurally distinct endobiotic and xenobiotic compounds, including pregnenolone, progesterone, lithocholic acid, paclitaxel, rifampicin, and the St. John’s wort constituent hyperforin (Bertilsson, G., et al. 1998; Kliewer, S.A., et al. 1998; Lehmann, J.M., et al. 1998; Moore, L.B., et al. 2000; Wentworth, J.M., et al. 2000). The activation of this xenobiotic sensor has also been linked to clinically-relevant drug interactions. For example, in patients taking the unregulated herbal antidepressant St. John’s wort, which contains the potent PXR agonist hyperforin (Moore, L.B., et al. 2000; Wentworth, J.M., et al. 2000), the upregulation of drug metabolism genes has been observed to generate significant decreases in the serum levels of therapeutics including oral contraceptives, anti-viral compounds, and immunosuppressant (Ernst, E. 1999; Fugh-Berman, A. 2000; Piscitelli, S.C., et al. 2000; Ruschitzka, F., et al. 2000).

PXR is a member of the nuclear receptor (NR) superfamily of ligand-activated transcription factors, which includes receptors for estrogen, progesterone, retinoid and thyroid hormones as well as retinoids, cholesterol metabolites and vitamins. Many

nuclear receptors bind to dual DNA response elements of various arrangements as either homodimers or as heterodimers with the retinoid X receptor-alpha (RXRα) (Aranda, A., et al. 2001; Giguere, V. 1999). In the absence of activating ligand, NRs have been shown to associate with transcriptional corepressors, which down-regulate gene expression by a variety of mechanisms including histone deacetylation (Aranda, A., et al. 2001; Rosenfeld, M.G., et al. 2001). In response to an activating ligand, however, NRs interact with transcriptional coactivators that up-regulate target gene expression in part by histone acetylation and by facilitating the recruitment of the basal transcriptional machinery (Aranda, A., et al. 2001; Rosenfeld, M.G., et al. 2001). PXR functions as a heterodimer with RXRα and has been shown to bind to a variety of dual DNA response elements arranged as direct and everted repeats. Upon ligand activation, PXR recruits several of the p160-class of transcriptional coactivators, including the steroid receptor coactivator-1 (SRC-1) (Bertilsson, G., et al. 1998; Blumberg, B., et al. 1998; Kliewer, S.A., et al. 1998; Lehmann, J.M., et al. 1998). For its potent control of CYP3A4 expression, PXR has been shown to employ two DNA response elements, one proximal (bases -172 to -149) and one distal (bases -7836 to -7607) relative to the start site of transcription. Both are required for maximal induction of gene expression in response to ligands (Goodwin, B., et al. 1999). PXR has also been shown to regulate the expression of MDR1 and CYP isoform 2B6 by using a combination of proximal and distal DNA response elements (Geick, A., et al. 2001; Wang, H.B., et al. 2003).

The PXRs of known sequence contain a ~50-amino acid insert unique to members of the nuclear receptor superfamily. This region is located between helices 1 and 3 within the canonical NR LBD fold, and adds a novel helix 2 and two β-strands adjacent to

PXR’s ligand binding cavity. Numerous crystal structures of the human PXR LBD have also revealed that the novel β-turn-β motif of this insert extends the two- to three- stranded antiparallel β-sheet common to NRs to a five-stranded β-sheet in PXR (Chrencik, J.E., et al. 2005; Watkins, R.E., et al. 2001; Watkins, R.E., et al. 2003a; Watkins, R.E., et al. 2003b). It is the terminal β-strands in each of these β-sheets that associate in an antiparallel fashion to generate the PXR homodimer, which produces a ten-strand intermolecular antiparallel β-sheet (Figure 1.2A). No other nuclear receptor has been observed to homodimerize in this fashion. In this work, structural, biophysical and functional features of this PXR homodimer are examined. Using sedimentation equilibrium experiments, the PXR LBD is shown to form a homodimer in solution with a Kd of 4.5 μM. Key residues at the dimer interface are also mutated and shown to disrupt

formation of the PXR dimer, which significantly reduces transcriptional activity and coactivator recruitment without impacting other necessary receptor actions like RXRα, DNA and ligand binding. Taken together, the data presented suggest that the Trp-Zip- mediated PXR homodimer interface plays a potential role in receptor function.

Experimental Procedures

PXR Expression and Purification

Wild-type human PXR LBD (residues 130-434) was coexpressed with a fragment of SRC-1 (residues 623-710) in E. coli BL21 (DE3) and purified using nickel-affinity

Trp223Ala/Tyr225Ala PXR LBD double-mutant was generated using the QuikChange mutagenesis kit (Stratagene), and expressed under the same conditions as wild-type PXR, but formed inclusion bodies in E. coli. The inclusion body pellet was washed twice with buffer containing 0.5% Triton X-100, 20 mM Tris-Cl pH 7.5, 250 mM NaCl, 50 mM imidazole, and 5% glycerol. Following the Triton X-100 wash, the pellet was resuspended in 6 M guanidinium hydrochloride pH 7.5 with the addition of 10 mM β- mercaptoethanol (BME), and stirred at 4 °C for 30 min. The denatured protein was ultra- centrifuged at 28.8K rpm for 30 min, diluted 1:3 with buffer (20 mM Tris-Cl pH 7.5, 250 mM NaCl, 50 mM imidazole, 10 mM BME and 5% glycerol) and then refolded by dialysis against this buffer with four changes. The refolded mutant protein was then purified under the same conditions as wild-type PXR LBD. In preparation for analytical ultracentrifugation, protein samples were concentrated to ~2.0 mg / ml and dialyzed (1:1000 (v/v) protein to dialysate) overnight with two buffer changes. The dialysis buffer (20mM Tris-Cl pH 7.5, 250mM NaCl, 2.5 mM EDTA, 5mM BME and 5% glycerol) was used to dilute protein to relevant concentrations. The cholesterol drug SR12813 (Sigma) was added at a 4-fold molar excess.

Analytical Ultracentrifugation

Sedimentation equilibrium experiments were performed using a Beckman XL-A analytical ultracentrifuge equipped with scanning absorption optics. Equilibrium measurements were obtained at three different rotor speeds (9,000, 13,000 and 16,000 rpm) and three concentrations (8.6, 17.3 and 21.7 μM) for wild-type PXR LBD and Trp223Ala/Tyr225Ala PXR LBD in triplicate. Baseline absorbance offsets were

established by increasing the rotor speed to 45,000 rpm for 6 hrs. Sedimentation equilibrium data was analyzed using the Beckman XL-A/XL-I data Analysis Software Version 4.0 which uses a nonlinear curve fitting procedure to determine the weight- average monomer molecular weight M and the association constant Ka according to the

following equation: c_r =c_monre RTM( )(r r ) +K_a

(

)

⎤ ⎢ ⎢ ⎣ ⎡ − − ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ − − 2 0 2 2 0 2 0 2 2 0 1 2 2 2 , 1 2 , ρ ν ω ρ ν ω

where cr is the concentration at radial position r, cmon,r₀is the concentration of the

monomer at the reference radius r₀, ωis the angular velocity in radians per second, R is the universal gas constant (8.314 x 107 erg·mol-1·K-1), T is the temperature in Kelvin, M is the monomer molecular weight, ν is the partial specific volume, ρis the density of the solvent, and Ka is the association constant, and E is the baseline offset. The association

constant, Ka was converted to the dissociation constant Kd by the following equation:

ε b K K a d 2 =

where b is the path length (1.2 cm) and ε is the molar extinction coefficient (28,390 M-1 cm-1 for PXR LBD) determined using the program Protean TM.

Circular Dichroism Spectropolarimetry

To confirm that the Trp223Ala/Tyr225Ala double-mutant form of the PXR LBD was properly folded, circular dichroism spectropolarimetry (CD) was performed using an Applied Photophysics PiStar-180 CD spectropolarimeter. The ellipticity from 210-300 nm was measured for wild-type PXR LBD and for the Trp223Ala/Tyr225Ala PXR LBD double-mutant. Both proteins were at 0.2 mg ml-1 in 100 mM phosphate buffer, pH 7.8,

100 mM NaCl and 5% glycerol. To examine thermal melting temperatures, the temperature was ramped from 20 to 98 ˚C while monitoring the ellipticity at 222 nm. Plots of fraction denatured versus temperature were produced by defining the upper and lower temperature baselines as 0 and 100%, respectively. Melting temperatures (Tm’s)

were defined as the point at which 50% of the sample denatured. Trials were performed in triplicate, and Tm’s for individual runs were averaged and standard errors calculated.

Transient Transfection Assays

Mutations in full-length PXR were generated with the Stratagene QuikChange site directed mutagenesis kit according to the manufacturer's instructions. Transfections were performed as described previously (Goodwin, B., et al. 1999; Watkins, R.E., et al. 2001). Briefly, CV-1 cells were plated in 96-well plates in phenol red-free Dulbecco’s modified Eagle’s medium containing high glucose and supplemented with 10% charcoal/dextran treated fetal bovine serum (HyClone, Logan, UT). Transfection mixes contained 5 ng of receptor expression vector, 20 ng of reporter plasmid, 12 ng of β-actin SPAP as internal control, and 43 ng of carrier plasmid. Plasmids for wild-type and mutant forms of human PXR and for the XREM-CYP3A4-LUC reporter, containing the enhancer and promoter of the CYP3A4 gene driving Luciferase expression, were as previously described (Goodwin, B., et al. 1999). Transfections were performed with LipofectAMINE (Life Technologies, Inc., Grand Island, NY) essentially according to the manufacturer’s instructions. Drug dilutions of rifampicin (Sigma, St. Louis, MO) and SR12813 (synthesized in-house) were prepared in phenol red-free Dulbecco’s modified Eagle’s

medium/F-12 medium with 15 mM HEPES supplemented with 10% charcoal-stripped, delipidated calf serum (Sigma, St. Louis, MO) which had previously been heat- inactivated at 62°C for 35 minutes. Serial drug dilutions were performed in triplicate to generate 11-point concentration response curves. Cells were incubated for 24 hours in the presence of drugs, after which the medium was sampled and assayed for alkaline phosphatase activity. Luciferase reporter activity was measured using the LucLite assay system (Packard Instrument Co., Meriden, CT) and normalized to alkaline phosphatase activity. EC50 values were determined by standard methods.

Immunocytochemistry

CV-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% charcoal-stripped calf serum (Hyclone, Logan, UT). The day before transfection, cells were plated at 2 x 105 cells per well of a 6 well plate (Becton Dickinson, Franklin Lakes, NJ) on ethanol-washed glass cover slips (Fisher Scientific, Pittsburgh, PA). Transfection was carried out using Effectene (Qiagen, Valencia, CA) according to the manufacturer’s specifications, with plasmids expressing wild-type PXR, Trp223Ala/Tyr225Ala PXR, or carrier DNA. Transfection complexes were suspended in phenol red-free DMEM/F12 plus 10% charcoal-stripped, delipidated calf serum (Sigma, St. Louis, MO), and left in contact with the cells overnight. The medium was then replaced, drugs [10 μM rifampicin (Sigma, St. Louis, MO) or 2 μM SR12813 (synthesized in house)] or 0.1% DMSO (Sigma, St. Louis, MO) were added, and the incubation was continued for a further 6 hours. For immunofluorescent staining of exogenous proteins, cultures were placed on ice for 5 minutes, rinsed 3 times with cold

PBS, then the cover slips were immersed in ice-cold acetone for 5 minutes and air-dried. Nonspecific antibody binding was blocked by incubating for 10 minutes in PBS containing 10% normal donkey serum (Jackson Immunoresearch Labs, West Grove, PA). Goat anti-PXR (Santa Cruz Biotechnology, Santa Cruz, CA) was applied in blocking buffer for 1 hour at a dilution of 1:100. The secondary antibody, donkey anti-goat IgG labeled with ALEXA 488 (Molecular Probes, Eugene, OR), was also applied for one hour in blocking buffer, but at a dilution of 1:1,000. Cultures stained without primary antibody were also obtained. Cover slips were mounted in 90% glycerol (Sigma, St. Louis, MO), 10% PBS, 4% n-propyl gallate (Sigma, St. Louis, MO) and 0.2 μM Hoechst 33258 (Molecular Probes, Eugene, OR). Fluorescent images were obtained on a Zeiss Axiovert 100 TV inverted microscope with a 100x objective under oil immersion, using Zeiss Axiovision software (Carl Zeiss, Inc., Thornwood, NY).

Gel Mobility Shift Assays

Gel mobility shift assays were performed as described before (Honkakoski, P., et al. 1998). Full-length human wild-type PXR, double-mutant (Trp223Ala/Tyr225Ala) PXR, and RXRα proteins were synthesized using the TNT quick-coupled in vitro transcription/translation system (Promega). Probes NR3 and ER6 from CYP2B6 and CYP3A4, respectively, were labeled with [γ-32P]dATP and purified by Microspin G-25 columns (Amersham Biosciences). Typically, 10 μL of binding reactions contained 10 mM HEPES (pH 7.6), 0.5 mM dithiothreitol, 15% glycerol, 0.05% Nonidet P-40, 50 mM NaCl, 2 μg of poly(dI-dC), with 0.1, 0.5, or 1 μL of in vitro translated nuclear receptor protein, and 4 X 104 cpm of labeled probe. After incubation at room temperature for 10

min, reaction mixtures were resolved on 5% acrylamide gels in 1 X Tris-acetic acid, EDTA buffer at 180 V for 1.5 h. Afterward, gels were dried, and autoradiography was performed overnight at -70 ˚C.

Competition Ligand Binding Assay

Polylysine YiO imaging beads (Amersham, GE Healthcare) were coated with histidine-tagged WT PXR LBD or Trp223Ala/Tyr225Ala PXR LBD, by mixing for 60 minutes at room temperature, in Tris buffer pH 8.0. Non-specific binding sites were blocked with a ten-fold excess of BSA for an additional 60 minutes at room temperature. The bead/receptor mix was washed and reconstituted in fresh assay buffer (50 mM Tris pH 8.0 containing 10% glycerol, 200mM KCl, 50uM CHAPS, 0.1 mg/mL BSA and 2mM DTT). Biotin (0.1 mM) was added to the suspension and allowed to mix for a further 60 minutes. The blocked receptor bead-mix was centrifuged at 1000 x g for 10 minutes at 4°C. The supernatant was discarded and the receptor-bead pellet was re-suspended in the appropriate volume of assay buffer. [N-methyl-3H]-GW0438X (synthesized at GSK and custom labeled at Amersham Biosciences, UK) was added to this suspension to achieve a final concentration of 10 nM. The receptor/imaging bead/radioligand mix was added directly to test compounds in 384-well plates in a one step addition. Test compounds were prepared from powder stocks by dissolving in DMSO and then serially diluted for displacement curves. Displacement of 10 nM [N-methyl-3H]-GW0438 was measured in a Viewlux 1430 ultraHTS microplate imager (Perkin Elmer Wallac Inc). Non-specific binding was determined in the presence of 10 μM GW0438. A similar competitive ligand binding assay method is described elsewhere (Nichols, J.S., et al. 1998). Data analysis

was achieved using a 3 parameter fit, assuming a slope of 1. The data were calculated as pIC50’s, but because the ligand concentration was well below the Kd for the receptor, this

value was not different from the pKi. The pKi is the –log of Ki, the inhibitor

concentration at which 50% inhibition is observed. Ki is calculated from the IC50 using

the Cheng-Prusoff equation:

Ki = IC50 / 1+([L]/Kd)

where L = concentration of free radioligand used in the assay and Kd = dissociation

constant of the radioligand for the receptor .

Pull-Down Assays

Pull-down studies were performed using a Profound Pull-Down kit (Promega) at 4 °C according to the manufacturer’s instructions. For the PXR-RXRα interaction, the RXRα-LBD (residues 225-462) was cloned into the pMALCH10T vector, expressed in E.coli BL21pLysS cells and purified by nickel affinity chromatography as described elsewhere (Ortlund, E.A., et al. 2005). Purified RXRα-LBD was biotinylated using an EZ-Link Sulfo-NHS-LC-Biotinylation kit (Promega) according to the manufacturer’s instructions. Biotinylated RXRα-LBD at 0.2 mg / mL was immobilized on streptavidin- agarose beads. The beads were washed with TBS buffer (25 mM Tris-Cl pH 7.0, 150mM NaCl), then blocked with biotin solution. The beads were equilibrated with binding buffer (50mM Tris-Cl pH 7.8, 250mM NaCl, 2.5 mM EDTA, and 5% glycerol) and wild- type (WT) PXR LBD or Trp223Ala/Tyr225Ala PXR LBD at 0.2 mg / mL was added to the beads and incubated for 12 hrs. Following prey capture, the beads were washed with binding buffer, eluted with elution buffer at pH 2.8, and examined by SDS-PAGE. For

the PXR-SRC-1 peptide interaction, biotinylated SRC-1 or random peptides at 0.2 mg / mL were immobilized on streptavidin-agarose beads. The beads were washed with TBS buffer (25 mM Tris-Cl pH 7.0, 150mM NaCl), then blocked with biotin solution. The beads were equilibrated with binding buffer (50mM Tris-Cl pH 7.8, 250mM NaCl, 10% glycerol, 0.5% Triton X-100) and WT PXR LBD or Trp223Ala/Tyr225Ala PXR LBD at 0.2mg / mL was added to the beads and incubated for 12 hrs. Following prey capture, the beads were washed with binding buffer, eluted with buffer at pH 2.8, and examined by SDS-PAGE.

Mammalian Two-Hybrid Studies

HepG2 cells were cultured in minimal essential medium (MEM) (Invitrogen) containing 10% fetal bovine serum albumin supplemented with 0.1 mM non-essential amino acids and 1 mM sodium pyruvate. 1000 ng of VP16_PXR (full-length WT PXR or Trp223Ala/Tyr225Ala PXR), 1000 ng of pM_SRC-1 NRID (nuclear receptor interaction domain I; SRC-1 residues 621-765), 900 ng 5xGal4Luc3 reporter plasmid (Chang, C., et al. 1999) and 100 ng pCMVB-gal (for normalization) were used for transfections